Pulmonary delivery offers a potential solution to the problem of developing an efficient, non-invasive administration route for proteins, peptides and other macromolecules. Considerable controversy exists, however, over both pathways involved in the alveolar epithelial absorption process and subsequent post-absorptive disposition of macromolecules. ^ A technique was developed for utilizing confocal laser scanning fluorescence microscopy (CLSFM) for examining peripheral regions of intact rat lung. It was then used in a comparative study of the pulmonary absorption and disposition of two fluoresceinated dextrans (3kDa (3KFD); 10kDa, (10KFD)), fluoresceinated insulin (FI) and 6-carboxyfluorescein (CF) following intratracheal administration to anesthetized rats. CF was cleared from peripheral airspaces within approximately one hour in a manner consistent with concurrent movement of probe along with aqueous solvent, perhaps through intercellular junctions or “gaps” in the alveolar epithelium. Both CF and 3KFD entered free interstitial spaces surrounding peripheral alveoli (filling and distending these regions), and were subsequently eliminated via capillaries, peripheral lymphatics, and the pleural membrane. The larger dextran (10kFD) remained almost entirely within the airspaces over the 2 hour experimental period. In contrast, FI was not seen within free interstitial spaces, nor did it enter peripheral lymphatics, suggesting a different post-absorptive disposition from that of the smaller molecules. Instead, FI was associated with septal and peripheral fibers, as well as other components of the extracellular matrix, which appeared as fluorescent sheaths beneath the epithelial surface. These results show that CLSFM represents a potentially powerful ancillary tool for gaining insight into macromolecular absorption and disposition processes within peripheral elements of the lung.